志贺菌ipaH基因PCR检测方法的建立及应用

Establishment and Application of PCR Detection Method for ipaH Gene of Shigella sp.

为了掌握青海省4种食源性动物体内志贺菌的分布情况,针对志贺菌属侵袭性质粒抗原H基因(invasive plasmid,ipaH)设计1对特异性引物,建立志贺菌的PCR检测方法,优化反应体系,检测该方法的敏感性和特异性,并将该方法应用于临床样本检测。结果表明,建立的PCR方法能特异性扩增志贺菌ipaH基因,而沙门氏菌、大肠杆菌、葡萄球菌、枯草杆菌、变形杆菌等均未扩增出条带;最低检测限值为1.8 pg/μL。在临床样本检测中,100株疑似菌株的阳性率为62.00%,其中牦牛源75.00%(3/4),鸡源35.29%(12/34),羊源44.44%(8/18),猪源88.64%(39/44);新鲜鸡粪和羊粪的阳性率分别为91.67%(11/12)和90.91%(10/11)。这说明试验所建立的PCR方法具有良好的敏感性和特异性,可应用于志贺菌的实验室诊断;4种动物体内均不同程度带菌,在临床诊断和治疗时不可忽视该菌的致病作用。

In order to grasp the distribution of 4 kinds of food-borne Shigella sp.in animals in Qinghai Province,a pair of specific primers were designed according to Shigella invasive plasmid antigen H gene(invasive plasmid,ipaH)to establish PCR detection method for Shigella sp..The reaction system was optimized,and the sensitivity and specificity of the method were detected.And this method was applied to detect the clinical samples.The results showed that the PCR method established by the experiment could specifically amplify the ipaH gene of Shigella sp.,but Salmonella sp.,Escherichia coli,Staphylococcus sp.,Bacillus subtilis,Proteus vulgaris,etc.did not amplify the specific band.The minimum detection limit was 1.8 pg/μL.Among the detected clinical samples,the positive rate of 100 suspected strains was 62.00%.And the positive rate of yak-borne,chicken-borne,sheep-borne,pig-borne Shigella sp.were 75.00%(3/4),35.29%(12/34),44.44%(8/18)and 88.64%(39/44)respectively.The positive rates of fresh chicken manure and sheep manure were 91.67%(11/12)and 90.91%(10/11),respectively.The results showed that PCR method established by the experiment had good sensitivity and specificity,and the method could be applied in the laboratory diagnosis of Shigella sp..The four kinds of animals carried pathogen in different degrees,so the pathogenic role of Shigella sp.could not be ignored in clinical diagnosis and treatment.